Various imaging systems and techniques have been used for biomedical applications, such as tissue mapping. One approach to tissue mapping involves multispectral imaging, where the tissue to be mapped is conjugate with a detector array, such as charge coupled device (CCD) or a photo-diode array. In the array, the spatial data is directly obtained in the image. A tunable filter, such a liquid crystal filter (LCTF) or acousto-optical tunable filter (AOTF) can be placed before the detector array, and images collected for each color setting of the filter. Alternatively, a volume hologram can be placed before the detector array, with images collected by pixel arrays positioned where each color is diffracted.
The above imaging systems, however, are generally limited in terms of temporal response time, typically about 25 microseconds (for the AOTF) to 100 milliseconds (for the LCTF) per wavelength. For weak intrinsic fluorescence, these later systems also have long array integration times, leading to multispectral-image acquisition times of several seconds—which are too long for live biological samples. Other approaches use confocal microscopy where high resolution images are obtained using a condenser lens to focus illuminating light from a point source into a diffracted limited spot within a specimen. An objective lens then focuses the light emitted from that spot onto a small pinhole and a detector measures the amount of light passing through the pinhole. Because only light from within the illuminated spot is focused through the pinhole, any stray light is filtered out, greatly improving the image quality. A coherent image is created by scanning point by point over the desired field of view, and recording the intensity of the light emitted from each spot, as small spots are illuminated at any one time. Scanning can be accomplished in several ways, such as laser scanning.
Multi-spectral confocal mapping holds great promise for imaging cancer cells at the cellular and molecular levels. Indeed, the National Institute of Health (NIH) has encouraged research and development pertaining to in-vivo image guided cancer interventions. Also, many cancers can be detected with multi-spectral imaging of fluorescence. See Thiberville, L., Moreno-Swirc, S., Vercauteren, T., Peltier, E., Cave, C., Heckly, G. B., “In Vivo Imaging of The Bronchial Wall Microstructure Using Fibered Confocal Fluorescence Microscopy,” Am J Respir Crit Care Med 175, 22-31 (2007); Fu, S., Chia, T. C., Kwek, L. C., Diong, C. H., Tang, C. L., Choen, F. S., Krishnan, S. M., “Application of Laser Induced Autofluorescence Spectra Detection In Human Colorectal Cancer Screening,” Proc. SPIE-OSA Biomedical Optics 5141, 298-304 (2003); and Ramanujan, V. K., Ren, S., Park, S., Farkas, D. L., “Non-Invasive, Contrast-Enhanced Spectral Imaging of Breast Cancer Signatures In Preclinical Animal Models In Vivo,” J Cel Sci Therapy 1, 102-106 (2010).
Moreover, spectroscopic tools can provide multi-spectral data during laser excitation of fluorescence, 2-photon, and Raman spectroscopies, especially useful where samples are labeled with a variety of molecular-specific contrast agents. Multi-spectral imaging is typically done with one grating and multiple pixels in a detector array, but this approach is limited by read-out noise and dark current. As such, most biomedical applications center on labeled tissue and/or the use of costly PMT arrays, preventing clinical applications in humans. This latter limitation is addressed in U.S. Pat. No. 7,366,365 entitled “Tissue Scanning Apparatus and Method.” In this latter patent, there is described a system for multispectral confocal mapping of tissue suitable for scanning in-vivo or ex-vivo tissues where spectra can be acquired for each pixel in a confocal spatial scan. More particularly, there is described a confocal spatial scan system in conjunction with a fast fiber Bragg grating spectrometer. This combination maps wavelengths into time slots and is fiber based. The fiber that connects the fast optical spectrum analyzer to the scanner functions as the pinhole in a confocal microscope. That is, the cleaved end of fiber provides the confocal pinhole. The spectrum analyzer provides spectra derived from un-blazed fiber Bragg gratings that have delay lines between the gratings. The use of a single detector in a confocal arrangement easily offers spectral resolutions of about 1-2 nm.
Unfortunately, this latter approach above cannot offer spectral resolution above 10 nm in a practical and cost effective manner, a regime particularly useful for applications in humans such as optical biopsy and surgical margin assessments. Moreover, the fiber Bragg gratings in the '365 patent are realized using single mode fiber typically having a core diameter of about 3 μm, thereby limiting the collection efficiency, impractical for weak intrinsic fluorescence.